The tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) have shown anti-tumor activity in the treatment of non-small cell lung cancer (NSCLC). Dramatic and durable responses have occurred in NSCLC tumors with mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). In contrast, these inhibitors have shown limited efficacy in glioblastoma, where a distinct EGFR mutation, the variant III (vIII) in-frame deletion of exons 2-7, is commonly found. In this study, we determined that EGFRvIII mutation was present in 5% (3͞56) of analyzed human lung squamous cell carcinoma (SCC) but was not present in human lung adenocarcinoma (0͞123). We analyzed the role of the EGFRvIII mutation in lung tumorigenesis and its response to tyrosine kinase inhibition. Tissue-specific expression of EGFRvIII in the murine lung led to the development of NSCLC. Most importantly, these lung tumors depend on EGFRvIII expression for maintenance. Treatment with an irreversible EGFR inhibitor, HKI-272, dramatically reduced the size of these EGFRvIII-driven murine tumors in 1 week. Similarly, Ba͞F3 cells transformed with the EGFRvIII mutant were relatively resistant to gefitinib and erlotinib in vitro but proved sensitive to HKI-272. These findings suggest a therapeutic strategy for cancers harboring the EGFRvIII mutation.irreversible inhibitor ͉ lung cancer ͉ lung squamous cell carcinoma E pidermal growth factor receptor (EGFR) is commonly overexpressed and mutated in many human malignancies and is often associated with aggressive phenotypes (1-3). Before the recent discovery of the somatic mutations in the EGFR kinase domain (4-8) in non-small cell lung cancers (NSCLC), deletions of the extracellular domain were considered the most frequent EGFR mutations in the different tumor types (9-13). These deletions have an activating effect on the receptor, giving cells expressing these truncated receptors a proliferative advantage. The most common truncated receptor is the variant III EGFR deletion mutant (EGFRvIII, delta 801EGFR, del2-7 EGFR), containing an inframe deletion of exons 2-7 (801 bp) from the extracellular domain, initially characterized at the genomic level in glioblastoma.Studies using immunohistochemical assays with EGFRvIII mutant-specific antibodies suggest that this mutation is present in multiple other tumor types, including NSCLC (10,11,14). However, because of EGFR's large and complex genomic structure (28 exons spanning Ϸ190 kb) and its large intron 1 (123 kb) where genomic deletions frequently occur, it has been difficult to assess and verify the existence of the EGFRvIII mutations at the genomic level. EGFRvIII mutations have been well demonstrated in glioblastoma, where they are present in Ͼ50% of glioblastomas with amplification of EGFR gene locus (12, 15, 16), but no genomic evidence for the existence of EGFRvIII mutations has been reported in NSCLC. Furthermore, the role of EGFRvIII mutation in the potential pathogenesis of NSCLC is unclear.Here, we report that the EGFRvIII mut...

Telomeres are key structural elements for the protection and maintenance of linear chromosomes, and they function to prevent recognition of chromosomal ends as DNA double-stranded breaks. Loss of telomere capping function brought about by telomerase deficiency and gradual erosion of telomere ends or by experimental disruption of higher-order telomere structure culminates in the fusion of defective telomeres and/or the activation of DNA damage checkpoints. Previous work has implicated the nonhomologous end-joining (NHEJ) DNA repair pathway as a critical mediator of these biological processes. Here, employing the telomerasedeficient mouse model, we tested whether the NHEJ component DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was required for fusion of eroded/dysfunctional telomere ends and the telomere checkpoint responses. In late-generation mTerc ؊/؊ DNA-PKcs ؊/؊ cells and tissues, chromosomal end-to-end fusions and anaphase bridges were readily evident. Notably, nullizygosity for DNA Ligase4 (Lig4)-an additional crucial NHEJ component-was also permissive for chromosome fusions in mTerc ؊/؊ cells, indicating that, in contrast to results seen with experimental disruption of telomere structure, telomere dysfunction in the context of gradual telomere erosion can engage additional DNA repair pathways. Furthermore, we found that DNAPKcs deficiency does not reduce apoptosis, tissue atrophy, or p53 activation in late-generation mTerc ؊/؊ tissues but rather moderately exacerbates germ cell apoptosis and testicular degeneration. Thus, our studies indicate that the NHEJ components, DNA-PKcs and Lig4, are not required for fusion of critically shortened telomeric ends and that DNA-PKcs is not required for sensing and executing the telomere checkpoint response, findings consistent with the consensus view of the limited role of DNA-PKcs in DNA damage signaling in general.Telomeres are key structural elements for the protection and maintenance of linear chromosomes that are comprised of a series of repetitive DNA sequence elements (TTAGGG in humans) and an array of sequence-specific and -nonspecific DNA binding proteins that create a higher-order chromatin structure (21). Telomeres serve various cellular functions, including the prevention of recognition of the linear end as a DNA double-strand break (DSB) (20,49,85); the modulation of local chromatin structure, which influences the expression of those genes proximal to chromosome ends (5, 66, 67); and the regulation of telomere maintenance enzymes and their accessibility (21). The last function is crucial, given the end replication problem that plagues conventional DNA synthesis (65,90). Telomere sequences can be added by telomerase, a specialized reverse transcriptase that adds telomere repeat DNA to the ends of chromosomes, thereby restoring sequences lost after RNA primer removal and end processing following DNA replication (7,53). Human cells in culture that divide in the absence of telomerase experience telomere attrition on the order of 50 to 100 bp per ce...

Reactive oxygen species (ROS) have been found to trigger apoptosis in tumor cells. At the same time, telomerase is found to be associated with malignancy and reduced apoptosis. However little is known about the linkage between ROS such as OH and telomerase/telomere. To address the interrelations between OH and telomerase/telomere in tumor cell killing, HeLa, 293 and MW451 cells were induced to undergo apoptosis with OH radicals generated via Fe 2+ -mediated Fenton reactions (0.1 mM FeSO 4 plus 0.3^0.9 mM H 2 O 2 ) and telomerase activity, telomere length were measured during apoptosis. We found that during OH-induced apoptosis, telomere shortening took place while no changes in telomerase activity were observed. Our results suggest that OH-induced telomere shortening is not through telomerase inhibition but possibly a direct effect of OH on telomeres themselves indicating that telomere shortening but not telomerase inhibition is the primary event during OH-induced apoptosis. Strikingly, we also found that OH-induced apoptosis in HeLa cells is caspase-3-independent but is associated with reduction of mitochondrial transmembrane potential. Our results indicate that OH triggers apoptotic tumor cell death through a telomere-related, caspase-independent pathway. ß

Purpose The cornea is a main barrier to drug penetration after topical application. The aim of this study was to evaluate the abilities of micelles generated from a positively charged triblock copolymer to penetrate the cornea after topical application. Methods The triblock copolymer poly(ethylene glycol)-poly(ε-caprolactone)- g -polyethyleneimine was synthesized, and the physicochemical properties of the self-assembled polymeric micelles were investigated, including hydrodynamic size, zeta potential, morphology, drug-loading content, drug-loading efficiency, and in vitro drug release. Using fluorescein diacetate as a model drug, the penetration capabilities of the polymeric micelles were monitored in vivo using a two-photon scanning fluorescence microscopy on murine corneas after topical application. Results The polymer was successfully synthesized and confirmed using nuclear magnetic resonance and Fourier transform infrared. The polymeric micelles had an average particle size of 28 nm, a zeta potential of approximately +12 mV, and a spherical morphology. The drug-loading efficiency and drug-loading content were 75.37% and 3.47%, respectively, which indicates that the polymeric micelles possess a high drug-loading capacity. The polymeric micelles also exhibited controlled-release behavior in vitro. Compared to the control, the positively charged polymeric micelles significantly penetrated through the cornea. Conclusion Positively charged micelles generated from a triblock copolymer are a promising vehicle for the topical delivery of hydrophobic agents in ocular applications.

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